The main goal of these studies is to elucidate the mechanisms underlying voltage- and intracellular pHi- dependent gating of connexin(Cx)-based gap junction (GJ) channels and unapposed hemi channels (uHCs). pHi is a fundamental modulator of cell function that influences various physiological processes such as metabolism, proliferation, function of membrane channels and transporters, cell movement and contractility. pHi can change considerably during pathological processes, most often during ischemia, and H+ ions have been shown to have broad effects on electrical and metabolic cell-cell communication through GJs and paracrine signaling through uHCs. Sp. Aim 1 focuses on pHi-dependent modulation of gating by transjunctional voltage (Vj) in homotypic GJs. We have shown that each hemi channel within a GJ channel has two distinct gating mechanisms, termed fast and slow gates, that are sensitive to Vj and distinguished by the channel closure to a substate and fully, respectively. We will test the hypothesis that dynamic pHi-mediated changes in gj with modest acidification occur through modulation of the Vj sensitivity of the fast gate in a Cx- type dependent manner, while stronger acidification leads to full uncoupling in all Cxs due to the closure of the slow gate without changes in the sensitivity to Vj. We will test the hypothesis that acidification-mediated full uncoupling is due to transition of the slow gate from a closed to a deep-closed state that can be accelerated by applied Vjs and by chemical uncouplers. Sp. Aim 2 focuses on pHi-dependent modulation of Vj-gating in heterotypic GJs formed in tissues co-expressing several Cx isoforms. In heterotypic GJs, acidification-induced uncoupling is defined mainly by the Cx exhibiting higher sensitivity to pHi allowing to test at higher resolution than in homotypic GJs that indeed pHi-dependent gating is hemi channel-based and that fast and slow gates play different roles in pHi-dependent regulation of cell-cell coupling. We will test the hypothesis that the NT-M1 domain of Cx is directly involved in Vj- and pHi-dependent gating as suggested by our data from Cx43*mCx30.2 chimeras. Sp. Aim 3 focuses on pHi-dependent gating of uHCs. We will test the hypothesis that pH-dependent modulation of Vj-gating observed in GJ channels has a common background with voltage- gating of uHCs, i.e., alkalization reduces and acidification increases voltage-sensitive gating of uHCs. We will identify residues in the NT-M1 domain that affect unitary conductance, sensitivity to voltage and permeability to dyes of uHCs. We will test the hypothesis that the pH sensor of uHCs is on the cytoplasmic side as we have reported earlier for Cx46 uHCs. Comparison of Vj- and pHi-dependent gating properties among uHCs and corresponding GJ channels will advance our knowledge as to what extent docking of uHCs alters biophysical properties of GJs. In Sp. Aims 1 and 3, we will test whether Vj- and pH-dependent gating is associated with a change in calmodulin (CaM) co-localization with GJs and whether CaM influences the interaction between H+ ions and gating elements of slow and fast gates of GJs and uHCs. PUBLIC HEALTH RELEVANCE: The main goal of these studies is to elucidate the mechanisms underlying voltage- and intracellular pH-dependent gating of connexin(Cx)-based gap junction (cell-cell) channels and non-junctional hemi channels expressed on the cell surface, which mediate electrical and metabolic cell-cell communication and paracrine signaling. pH is a fundamental modulator of cell function that influences various physiological processes such as metabolism, proliferation, function of membrane channels and transporters, cell movement and contractility. The study of these mechanisms will help to identify new therapeutic approaches to treat ischemia and other diseases during which changes in pH take place and typically increase the severity of the pathological process.